Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells.

Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of >or=30 AP(+) cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable, and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.

Optic disc swelling and vision loss in a patient with cystic fibrosis and diabetes.

Advances in cystic fibrosis management have significantly improved life expectancy in these patients. However, we are now faced with a growing number of long-term extrapulmonary consequences of this disease, including ophthalmic complications of diabetes in cystic fibrosis patients. We present a unique report that documents a case of diabetic papillopathy progressing to nonarteritic anterior ischemic optic neuropathy resulting in vision loss in a patient with CF and diabetes. It highlights the potentially devastating consequences of longstanding diabetes in CF patients.

Massive colchicine overdose with recovery.

Colchicine is an active alkaloid that is commonly used for treatment of multiple diseases including gout, primary biliary cirrhosis and familial Mediterranean fever. Less commonly, it has been implicated in several fatal overdoses. Deaths from colchicine overdoses are usually due to multi-organ failure, whether directly from colchicine toxicity or due to ensuing sepsis. We report an extreme case of colchicine ingestion (1.38 mg/kg), which is the largest reported non-fatal colchicine overdose. The patient was a 47-year-old First Nations woman with a history of depression and no other comorbidities. Ingestion was intentional and initial presentation was within 2 h of ingestion, at which point she had normal clinical and laboratory parameters. Early implementation of a targeted therapeutic strategy directed at the predicted multi-organ failure which included aggressive use of a GI decontamination protocol, timely supportive measures including ventilator support and renal replacement therapy, as well as the utilization of broad-spectrum antibiotics and G-CSF for sepsis and leucopenia management, resulted in successful support and discharge of this patient off dialysis.